58400 - Protocol (50 prep)
Contents
1. Slurry E 14 5 mL ExoC Buffer 8 mL ExoR Buffer 12 mL Lysis Buffer A 20 mL Lysis Additive B 2mL Wash Solution A 18 mL Elution Solution A 6 mL Mini Filter Spin Column 50 Mini Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 100 Product Insert 1 Please check page 4 for Average Yields and Common RNA Quantification Methods Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25 C for up to 2 year without showing any reduction in performance It is recommended to warm Lysis Buffer A for 20 minutes at 60 C if any salt precipitation is observed Important Note gt Urine samples stored at 70 C 20 C or at 4 C will develop some precipitation due to the aggregation of some of the highly abundant proteins in urine Eliminating these precipitates using centrifugation or filtration may cause the loss of exosomes Furthermore these precipitates may affect the quality of the purified nucleic acid We recommend the use of Norgen s Urine Preservative when collecting urine samples which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures The components of the Urine Preservative allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA RNA or proteins Norgen s Urine Preservative is available as a liquid format in Norgen s Urine Preservative Single Do2. encapsulated within exosomes or bound to proteins fc RNA are protected from degradation by RNAses they can be efficiently recovered from biological fluids such as urine In general these two RNA groups contain valuable information for the discovery of biomarkers that can help with early detection of certain cancer types and for monitoring the disease status Norgen s Exosome Purification and RNA Isolation Mini Kit constitutes an all in one system for the purification of exosomes and the subsequent isolation of exosomal RNA from different urine sample volumes ranging from 250 uL to 1 mL The purification is based on spin column chromatography that employs Norgen s proprietary resin The kit is designed to isolate all sizes of extracellular vesicle RNA including microRNA The kit provides a clear advantage over other available kits in that it does not require any special instrumentation protein precipitation reagents extension tubes phenol chloroform or protease treatments Moreover the kit allows the user to elute into a flexible elution volume ranging from 50 uL to 100 uL The purified RNA is free from any protein bound circulating RNA and of the highest integrity The purified RNA can be used in a number of downstream applications including real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays Component Product 58400 50 preps
3. fresh 1 7 mL Elution tube Apply 50 uL of Elution Solution A to the column and centrifuge for 1 minute at 2 000 RPM followed by 2 minutes at 8 000 RPM 10 For maximum recovery transfer the eluted buffer back to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 400 x g 2 000 RPM followed by 2 minutes at 5 800 x g 8 000 RPM gt Exosomal RNA is now ready for downstream applications Appendix A Exosomal RNA Yield Exosomal RNA like RNA in other cell free bodily fluids is normally found in very low amounts 1 100 pg uL therefore measuring exosomal RNA concentration using common quantification methods is very difficult and challenging Typical yields of exosomal RNA vary significantly from sample to sample Variability is also observed between samples collected from the same donor at different times during the day and therefore there is no absolute yield for RNA purified from bodily fluids Exosomal RNA yield varies depending on a number of factors including age sex diet exercise and most importantly the health status of the donor Below is a list of the most common RNA quantification methods as well as the limit of detection for each of these methods Unfortunately none of these methods can be used reliably for measuring the concentration of RNA purified from exosomes unless large volumes have been processed This would only be applicable if exosomes contains the maximum amount of RNA tha
4. general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 or call one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2015 Norgen Biotek Corp PI58400 1
5. gt After incubation mix well by vortexing for 10 seconds then centrifuge for 2 minute at 500 RPM Transfer the supernatant to a Mini Filter Spin column assembled in an elution tube and centrifuge for 1 minute at 6 000 RPM Do Not Discard the flowthrough which contains your purified Exosomes Your Exosomes are now ready for RNA Isolation Section 2 or any other downstream applications Section 2 Exosomal RNA Isolation 1 Add 300 uL of Lysis Buffer A and 37 5 uL of Lysis Additive B to the 200 uL ExoR Buffer containing the purified Exosomes Section 1 Step 7 2 Mix well by vortexing for 10 seconds then incubate at room temperature for 10 minutes 3 After incubation add 500 uL of 96 100 Ethanol to the mixture from Step 2 and mix well by vortexing for 10 seconds 4 Transfer 500 uL of the mixture from Step 3 into a Mini Spin Column Centrifuge for 1 minute at 3 300 X g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 5 Repeat Step 4 one more time to transfer the remaining mixture from Step 3 into the Mini Spin Column 6 Apply 600 uL of Wash Solution A to the column and centrifuge for 30 seconds at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 7 Repeat step 6 one more time for a total of two washes Spin the column empty for 1 minute at 13 000 x g 14 000 RPM Discard the collection tube 9 Transfer the spin column to a
6. gt k 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com Urine Exosome Purification and RNA Isolation Mini Kit Product Insert Product 58400 Exosomes are 40 150 nm membrane vesicles which are secreted by most cell types Exosomes can be found in plasma serum saliva urine amniotic fluid and malignant ascite fluids among other biological fluids Evidence has been accumulating recently that these vesicles act as cellular messengers conveying information to distant cells and tissues within the body The exosomes contain cell specific proteins lipids and RNAs which are transported to other cells where they can alter function and or physiology These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours tissue repair neural communication and transfer of pathogenic proteins Recent work has demonstrated the presence of distinct subsets of microRNAs within exosomes and other extracellular vesicles EVs which depend upon the tumour cell type from which they are secreted For this reason exosomal RNA may serve as biomarkers for various diseases including cancer Another subset of RNA that is found in urine are the free circulating RNA fc RNA These fc RNA are usually protein bound RNA that are leaked from cells either during apoptosis or necrosis As the RNA molecules
7. www norgenbiotek com Lysis Buffer A contains guanidine thiocyanate and should be handled with care Guanidine thiocyanate forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of this solution If liquid containing these buffers is spilled clean with suitable laboratory detergent and water If the spilled liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite Important Notes Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defence against these enzymes e The RNA area should be located away from microbiological work stations Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination There should be designated solutions tips tubes lab coats pipettes etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions Whe
8. This varies from individual to individual based on numerous variables In order to increase the yield the amount of urine plasma or serum input could be increased 7 Why is the A260 280 ratio of the purified RNA lower than 2 0 e Most of the exosomal RNA is short RNA fragments with a very low concentration where the A260 280 ratio tends to decrease with the decrease in the RNA concentration The A260 280 ratio is normally between 1 1 6 This low A260 280 ratio will not affect any downstream application 8 Why does my isolated RNA not perform well in downstream applications e lf a different Elution Buffer was used other than the one provided in the kit the buffer should be checked for any components that may interfere with the application Common components that are known to interfere are high salts including EDTA detergents and other denaturants Check the compatibility of your elution buffer with the intended use 9 What should do if some of the grey resin is transferred out when am decanting the urine supernatant e Simply remix and recentrifuge After centrifuging decant the supernatant 10 What if added more or less of Slurry E e Adding less volume may reduce the amount of the purified exosomes Adding more may not affect the exosome capture but ma affect the release of the purified exosomes in the ExoR Buffer 11 What if added more or less of ExoC Buffer e Adding a different volume from the specified optimum volume wi
9. dard Curve lg alia aan SEE a gia hen a a a pp 1 0 Log Starting Quantity O Standard gt lt Unknown SYBR E 296 1 R 2 0 890 Slope 1 673 y int 32 707 Frequently Asked Questions 1 What If a variable speed centrifuge is not available e A fixed speed centrifuge can be used however reduced yields may be observed 2 At what temperature should centrifuge my samples e All centrifugation steps are performed at room temperature Centrifugation at 4 C will not adversely affect kit performance 3 What if added more or less of the specified reagents volume e Adding more or less than the specified volumes may reduce both the quality and the quantity of the purified RNA Eluting your RNA in high volumes will increase the yield but will lower the concentration Eluting in small volumes will increase the concentration but will lower the overall yield 4 What if I forgot to do a dry spin before my final elution step e Your purified RNA will be contaminated with the Wash Solution A This may reduce the quality of your purified RNA and will interfere with your downstream applications 5 Can I perform a second elution e Yes but it is recommended that the 2 elution be in a smaller volume 50 of 1 Elution It is also recommended to perform the 2 elution into a separate elution tube to avoid diluting the 1 elution 6 Why do my samples show low RNA yield e Exosomes contain very little RNA
10. ll signficiantly reduce the amount of the purified exosomes 12 What if added more or less of ExoR Buffer e Adding less volume will reduce the release of the captured exosomes in the ExoR Buffer Adding more will not affect the release of the captured exosomes but it will be more diluted 13 What will happen if accidently some of the grey resin was transferred with the ExoR buffer e Any grey resin will be filtered through the Mini Filter Spin Column and the flowthrough which contains the purified exosomes should not contain any grey resin Related Products Product Urine Collection and Preservation Tubes 50 cc 1 tube 18111 Urine Collection and Preservation Tubes 50 cc 50 tubes 18113 Urine Collection and Preservation Tubes 15 cc 1 tube 18120 Urine Collection and Preservation Tubes 15 cc 50 tubes 18122 Urine Collection and Preservation Tubes 5 cc 1 tube 18116 Urine Collection and Preservation Tubes 5 cc 50 tubes 18118 Urine Preservative Single Dose 1 tube 18124 Urine Preservative Single Dose 50 tubes 18126 Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Exosome Purification and RNA Isolation Mini Kit or NORGEN products in
11. n working with purified RNA ensure that they remain on ice during downstream applications Notes Prior to Use VVV VVV Vv All centrifugation steps are performed at room temperature Ensure that centrifuge tubes used are capable of withstanding the centrifugal forces required The provided spin columns are optimized to be used with a benchtop centrifuges and not to be used on a vacuum apparatus Most standard benchtop microcentrifuges will accommodate Norgen s Micro Spin Columns Centrifuging Norgen s Spin Columns at a speed higher than recommended may affect RNA yield Centrifuging Norgen s Spin Columns at a speed lower than recommended will not affect RNA yield However centrifugation at a lower speed may require longer time for the solutions to pass through the spin column Ensure that all solutions are at room temperature prior to use It is highly recommended to warm up Lysis Buffer A at 60 C for 20 minutes and mix well until the solutions become clear again if precipitates are present Prepare a working concentration of the Wash Solution A by adding 42 mL of 96 100 ethanol provided by the user to the supplied bottle containing 18 mL of concentrated Wash Solution A This will give a final volume of 60 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added If any of the solutions do not go through the Spin Columns within the specified centrifugation time spin for an additional 1 2 minu
12. se Ampules as well as in a dried format in Norgen s Urine Collection and Preservation Tubes please see Related Products Table Customer Supplied Reagents and Equipment e Disposable powder free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters Vortex 96 100 Ethanol Nuclease Free Water General Precautions All biological samples should be considered as potentially infectious Proper biosafety measures should therefore be carried out when using this kit Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Urine Exosome Purification and RNA Isolation Mini Kit is tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine Exosome Purification and RNA Isolation Mini Kit is designed for research purposes only It is not intended for human or diagnostic use Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use Safety Information Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at
13. t can fit within the specification range of these quantification tools It should be noted that the specifications outlined below are based on measuring a pure RNA sample which will not be the case for the exosomal RNA purified from urine Exosomal RNA is short fragmented RNA which is usually present in less than 1000 bp Purified exosomal RNA usually contains traces of proteins which will interfere with most quantification methods leading to the overestimation of the purified RNA concentration Therefore purified RNA contaminated with more proteins will be presented at a higher concentration as compared to RNA purified with less protein contaminants which in this case will depend on the method used for urine RNA purification The only reliable method that can assess the quality and the relative quantity of the purified plasma serum RNA is RT qPCR amplification of a standard RNA using a small RNA amplicon such as the 5S rRNA housekeeping gene Common RNA Quantification Methods 1 Bioanalyzer RNA Quantification kits Pl RNA 6000 Nano Kit RNA 6000 Pico Kit Small RNAkit 25 500 ng L 25 250ngpL 50 2000 pg L 50 5000 a 5000 Qualititative range 5 500 ng L 5 250 ng uL pg uL pg uL 50 2000 pg uL Quantitation F 7 S accuracy aiad Sak eee fee a 2 NanoDrop 2000 gt Detection Limit 2 ng ul dsDNA 3 Quant iT RiboGreen RNA Assay Kit gt Quantitation Range 1 200 ng 4 qPCR Standard Curve generated by Norgen Stan
14. tes until the solution completely passes through the Column Do NOT exceed the centrifugation speed as this may affect RNA yield Preparation of Cell free Urine Sample 1 Collect and transfer 15 50 mL of the urine into a conical tube and centrifuge at 200 x g 1 000 RPM for 10 minutes to remove urine exfoliated cells and debris Decant cell free urine into new 15 50 mL conical tube Centrifuge the cell free urine at 1 800 x g 3 000 RPM for 10 minutes to remove any residual debris or bacterial cells Transfer cell free urine into a fresh 15 50 mL conical tube Section 1 Exosome Purification from 250 uL 1 mL Cell Free Urine Note The procedure outlined below is for 1 mL input of urine If processing a sample volume lower than 1 mL urine simply bring the volume of your samples up to 1 mL using Nuclease free water and proceed as outlined below 1 NOOR WD To 1 mL urine add 100 uL of ExoC Buffer followed by the addition of 200 uL of Slurry E Note Mix Slurry E well prior to use For optimal performance ensure _that resin is completely resuspended Mix well by vortexing for 10 seconds and let stand at room temperature for 5 minutes Mix well by vortexing for 10 seconds Centrifuge for 2 minutes at 2 000 RPM Discard the supernatant Apply 200 uL ExoR Buffer to the slurry pellet and mix well by vortexing for 10 seconds Incubate the slurry pellet resuspended in the 200 uL ExoR Buffer at room temperature for 5 minutes
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